CN114591976A - Gene for encoding GA2 ox-oxidase and application thereof in judging dwarf mango variety - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a gene for encoding GA2 ox-oxidase and application thereof in judging a mango dwarfing variety. A gene for encoding GA2 ox-oxidase, wherein the nucleotide sequence of the gene is shown as SEQ ID No. 1. The GA2 ox-oxidase gene can provide support for early prejudging whether the test material has dwarfing characteristics.

Description

A gene encoding GA2ox-oxidase and its application in judging mango dwarf varieties

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a gene encoding GA2ox-oxidase and its application in judging dwarf mango varieties.

Background technique

Dwarf planting is a constant development trend of crop production. Plant dwarfing is mainly divided into genetic dwarfing and physiological dwarfing. A single gene mutation belongs to hereditary dwarfing, and its dwarfing incentive is mostly gene synthesis protein, protein affects hormones, and hormones affect phenotype; hand environmental factors lead to changes in plant phenotype, which belongs to physiological dwarfing, and its dwarfing effect The mechanism is mostly that environmental changes affect the secretion of plant hormones, which eventually lead to changes. However, no matter which dwarfing mode, the phenotype must be affected by hormones, and the hormones in the process of plant growth are mostly related to gibberellin.

There are many enzymes involved in the synthesis and decomposition of gibberellin in plants, and GA2ox (gibberellin 2β oxidase) is one of them. It is an enzyme that inhibits the synthesis of active gibberellin, which can reduce the content of active gibberellin, so as to achieve The purpose of regulating plant height. The regulation of GA metabolic pathways by GA2ox has been demonstrated in Arabidopsis.

By comparing the expression levels of GA2ox genes in plant materials with arborization characteristics and dwarf characteristics, it is possible to predict whether plants have dwarf characteristics, which saves time and effort.

SUMMARY OF THE INVENTION

In order to overcome the deficiencies of the above technologies, the present invention provides a gene encoding GA2ox-oxidase and its application in judging mango dwarf varieties, which can predict whether mango varieties have dwarf characteristics.

To achieve the above object, the present invention provides the following technical solutions:

The first object of the present invention is to provide a gene encoding GA2ox-oxidase, and the nucleotide sequence of the gene is shown in SEQ ID No.1.

The second object of the present invention is to provide the application of the gene encoding GA2ox-oxidase for predicting whether mango varieties have dwarfing characteristics.

The third object of the present invention is to provide a GA2ox-oxidase, the GA2ox-oxidase is encoded by the above-mentioned gene.

Preferably, the amino acid sequence of the GA2ox-oxidase is shown in SEQ ID No. 2; the GA2ox-oxidase consists of 341 amino acids, the molecular weight is 38729.40 Da, and the theoretical isoelectric point is 6.56.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The present invention obtains the GA2ox-oxidase gene sequence from the test material, analyzes the physicochemical activity of the gene, and uses the gene as an index for regulating dwarfing traits.

(2) The present invention involves the cloning of the GA2ox-oxidase gene and the analysis of the corresponding amino acid sequence. The semi-fluorescence quantitative method is used to determine the expression of the GA2ox-oxidase gene, which can be used to predict whether the test material has dwarfing in the early stage. traits are supported.

Description of drawings

Fig. 1 is the result of PCR amplification of the intermediate fragment of GA2ox-oxidase gene;

Figure 2 is a graph showing the results of PCR amplification of the 3' end of the GA2ox-oxidase gene;

Figure 3 is a graph showing the results of PCR amplification of the 5' end of the GA2ox-oxidase gene;

Figure 4-6 is a schematic diagram of the homology comparison results of mango GA2ox and other plant GA2ox;

Among them, Dz: durian; Hb: rubber tree; Hu: mallow; Jc: jatropha; Me: cassava; Mg: mango; Rc: castor; Tc: cocoa;

Figure 7 is an analysis diagram of the signal peptide and transmembrane domain of the GA2ox-encoded protein;

Figure 8 is an analysis diagram of the secondary structure of the protein encoded by GA2ox;

Fig. 9 is the hydrophobicity analysis diagram of GA2ox-encoded protein;

Figure 10 is a phylogenetic tree of mango GA2ox;

Figure 11 is a graph of GA2ox gene expression in Jinhuangmang and Guiqimang in different phenological stages.

Detailed ways

The technical solutions of the patent of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts shall fall within the protection scope of the present invention.

2000分光光度计：Thermo；恒温摇床；

超保真DNA聚合酶：NEB；FirstChoice RLM-RACEKit：Thermo；2×Taq Master Mix(Dye Plus)：Vazyme。In Example 1, the main instruments and reagents are: PCR instrument: Takara; Electrophoresis instrument: Bio-Rad;

2000 spectrophotometer: Thermo; constant temperature shaker;

Ultra-fidelity DNA polymerase: NEB; FirstChoice RLM-RACEKit: Thermo; 2 x Taq Master Mix (Dye Plus): Vazyme.

2000分光光度计：Thermo；普通PCR仪：Takara；All-in-One^TMFirst-Strand cDNA Synthesis Kit：GeneCopoeia(Cat.No.AORT-0050)；All-in-One^TMqPCR Mix：GeneCopoeia(Cat.No.AOPR-0200)；Epcentre^TM RNase R。In Example 2, the main instruments and reagents are: ABI 7900-Fast Real-Time PCR Detection System: ABI;

2000 Spectrophotometer: Thermo; General PCR Machine: Takara; All-in-One ^™ First-Strand cDNA Synthesis Kit: GeneCopoeia (Cat. No. AORT-0050); All-in-One ^™ qPCR Mix: GeneCopoeia (Cat. No. AOPR-0200); Epcentre ^™ RNase R.

Example 1 : Cloning and sequence analysis of mango GA2ox-oxidase gene

1. RNA extraction and extraction

1. Total RNA extraction (Trizol method)

(1) Sample processing: Take 80-100 mg of mango leaves into a freezing mortar, add liquid nitrogen to grind to powder, transfer to a 1.5 mL centrifuge tube containing 1 mL of Trizol, shake and mix well, and let stand at room temperature for about 5 minutes.

(2) Phase separation: add 0.2 mL of chloroform to each 1 mL of Trizol, shake and mix for 15 s, then stand at room temperature for about 3 min, 4° C., 12000 rpm, and centrifuge for 15 min.

(3) Precipitation: transfer the aqueous phase to a new 1.5 mL centrifuge tube, add 0.5 mL of isopropanol per 1 mL of Trizol, mix well, let stand at room temperature for 10 min, 4°C, 12000 rpm, and centrifuge for 10 min.

(4) Washing: discard the supernatant, add 1 mL of 75% ethanol to each 1 mL of Trizol, mix well, and centrifuge for 5 min at 4°C, 7500 rpm.

(5) Dissolution: Discard the supernatant, air dry the RNA precipitation for about 5 minutes (be careful not to dry it completely, only the precipitation is white), and add DEPC to treat the water to dissolve the RNA precipitation.

2. Purification of Extracted RNA

(1) Prepare the following reaction solution in a microcentrifuge tube, 50ul system:

(2) Recombinant DNase I was inactivated after 25 min of reaction at 37°C.

(3) Add 10ul 3M sodium acetate and 250ul cold ethanol, and place at -80°C for 20min.

(4) Centrifuge at 12,000 rpm for 10 min at 4°C, and discard the supernatant.

(5) Add 70% cold ethanol to wash, centrifuge at 4°C and 12000rpm for 5min, discard the supernatant, and dry the precipitate.

2. Cloning the intermediate fragment of the target gene

2.1 Design a pair of degenerate primers according to the conserved region according to the cDNA sequence of the plant GA2ox gene published in GenBank and after the DNAMAN software alignment.

GA2ox-F: AAYGGYGATR TBGGHTGGRTYGAATA

GA2ox-R: TGCYTYACRCTYTTA AAYCTYCCATTWGTCA

2.2 Reverse transcription of intermediate fragments

(1) Thaw the reagents of the First-Strand cDNA Synthesis Kit, mix by gently inverting, centrifuge briefly, and place on ice for later use.

(2) Prepare RNA-Primer Mix on ice, add the following reagents to a pre-cooled RNase-free reaction tube to a total volume of 13 μL;

(3) Mix the RNA-Primer Mix, centrifuge briefly, denature it at 65°C for 10 min, and place it on ice immediately;

(4) Add the following reagents to the RNA-Primer Mix reaction tube to a total volume of 25 μL;

(5) Mix the reaction mix, briefly centrifuge and incubate at 37°C for 1h;

(6) After the reaction, inactivate at 85°C for 5 min, and store the reverse transcription product at -20°C for later use.

2.3 Analysis and separation of intermediate fragments

Intermediate fragment PCR reaction system:

The PCR reaction program is: 94℃ for 5min; 94℃ for 40s, 58℃ for 40s, 72℃ for 1min, 35 cycles; 72℃ for 10min; 4℃ for 10min; Electrophoresis analysis, the results are shown in Figure 1, and gel cutting was performed to recover the target fragment.

As can be seen from Figure 1, the size of the GA2ox intermediate fragment cloned from mango leaves is about 900 bp.

2.4 Target fragment recovery

(1) Add 50 μL of the reaction system to the spotting well of 1% agarose gel, and spot DNA Marker DM 2000 on one side of the sample.

(2) Constant voltage 120V, electrophoresis for about 30min, according to the position of the band movement, judge when to terminate the gel running.

(3) Under the ultraviolet lamp, according to the position of the Marker, the target band is cut and recovered, and the full-length fragment of the target gene is purified by the DNA agarose recovery kit.

(4) Quickly put the cut gel into a centrifuge tube, and evaluate its volume according to the weight of the gel.

(5) Add the same volume of Binding Buffer to the gel at 65°C for 5 min until the gel is completely melted.

(6) Suction the thaw solution into the HiBind DNA Mini column, then put the column into a centrifuge tube, centrifuge at 10000g, room temperature for 1min. Discard the waste solution and leave the column until all the thaw solution has completed this step.

(7) Add 300 μL of XP2 to the column, 10000 g, room temperature, and centrifuge for 1 min. Discard the waste liquid and keep the column.

(8) Add 700 μL of SPW Wash buffer to the column, 10000 g, room temperature, centrifuge for 1 min, and discard the waste liquid. Repeat this step once.

(9) Room temperature, 13000g, idling for 2min, to remove residual waste liquid.

(10) Put the column into a new centrifuge tube, drop 30 μL of Elution Buffer into the center of the membrane, at room temperature for 2 minutes, and then centrifuge at 13000g for 2 minutes to elute the DNA.

2.5T vector ligation and transformation

I. Connection

4 μL of PCR product and 1 μL of PEASY-T1 cloning vector were mixed gently and reacted at room temperature for 5 min. After the reaction is complete, place the centrifuge tube on ice.

II. Transformation

(1) Take out the E. coli competent cell DH5a from the -80°C refrigerator, put it in ice until it is completely dissolved, and mix gently.

(2) Aspirate 50 μL of competent cells into a sterilized centrifuge tube, add 5 μL of recombinant plasmid to the centrifuge tube, mix gently, and let stand on ice for 30 min.

(3) Place the ice-bathed centrifuge tube in a 42°C water bath for heat shock for 90s, quickly move it into an ice box with ice for 2min, add 600ul LB culture medium, and place it on a 37°C constant temperature shaker for 1 hour at 220rpm.

(4) Take 50 ul of the culture solution and apply it to the solid LB medium containing Amp, and after drying, place it in an incubator at 37° C. for upside-down culture. After 12h-16h, check whether colonies appear in the petri dish.

(5) Pick a single clone for streak culture, and use the gene upstream and downstream primers for colony PCR amplification detection.

(6) Pick positive single clones for shaking and sequencing.

3. Cloning the 3' end of the target gene

3.1 Design a pair of 3'RACE primers according to the obtained intermediate fragment of the target gene.

GA2ox 3' GSP-1: CCGCCATGTCCGGATATTCA

GA2ox 3'GSP-2: ATCCAACAACACTTCCGGCT

3.2 Obtaining 3' cDNA

(1) Add the following components to an RNase-free centrifuge tube and operate on ice:

(2) Mix gently, incubate at 42°C for 1 h, and store at -20°C.

3.3 Outer 3' PCR reaction system

The PCR reaction program was: 94°C for 3 min; 94°C for 40s, 60°C for 40s, 72°C for 1 min, 35 cycles; 72°C for 10 min; 4°C for 10 min.

3.4 Inner 3' PCR reaction system

The PCR reaction program is: 94℃ for 3min; 94℃ for 40s, 60℃ for 40s, 72℃ for 1min, 35 cycles; 72℃ for 10min; 4℃ for 10min; Electrophoresis analysis, the results are shown in Figure 2, and the target fragment was recovered by gel cutting and connected to the PEASY-T1 cloning vector to transform competent cells, and sequencing was performed after the positive colonies were identified.

As can be seen from Figure 2, specific primers GA2ox 3'GSP-1, GA2ox 3'GSP-2 and their respective universal primers were designed according to the sequence information of the obtained GA2ox gene intermediate fragment for PCR amplification to obtain a DNA of about 900bp in size Fragment.

4. Cloning the 5' end of the target gene

4.1. Design a pair of 5'RACE primers according to the obtained intermediate fragment of the target gene.

GA2ox 5'GSP-1:GCACCTGCATAATCAACCACC;

GA2ox 5'GSP-2:GCTTGGGTACTGGCAAAAGC.

4.2 Outer 5'PCR reaction system

The PCR reaction program was: 94°C for 3 min; 94°C for 40s, 60°C for 40s, 72°C for 1min, 35 cycles; 72°C for 10min; 4°C for 10min;

4.3 Inner 5'PCR reaction system

The PCR reaction program is: 94℃ for 3min; 94℃ for 40s, 60℃ for 40s, 72℃ for 1min, 35 cycles; 72℃ for 10min; 4℃ for 10min; Electrophoresis analysis, the results are shown in Figure 3, and the target fragment was recovered by gel cutting and connected to the PEASY-T1 cloning vector to transform competent cells, and sequencing was performed after identifying positive colonies.

As can be seen from Figure 3, specific primers GA2ox 5'GSP-1, GA2ox 5'GSP-2 and their respective universal primers were designed according to the sequence information of the obtained GA2ox gene intermediate fragment to carry out PCR amplification to obtain DNA with a size of about 500bp Fragment.

5. Gene sequence splicing and analysis

5.1 Use DNAMAN to splicing the 5' end, 3' end and the middle fragment to obtain the full-length sequence of the GA2ox gene and translate it into a protein sequence; then use BLAST to perform amino acid similarity search, and the results are shown in Figure 4-6.

It can be seen from Figure 4-6 that the GA2ox sequence is 79% identical to orange (XM_006467404.3);

79% consistency with citrus (XM_006449629.2);

78% consistency with cassava (XM_021738345.1);

78% consistency with Hevea brasiliensis (XM_021820571.1);

The consistency with upland cotton (XM_016896568.1) is 76%;

The consistency with Chinese cotton (XM_012597405.1) is 75%.

5.2 Analysis of physicochemical properties of amino acid sequences

Using protparam (http://web.expasy.org/protparam/) analysis, the GA2ox sequence contains a complete open reading frame, and the stop codon is TGA, respectively. GA2ox encodes 341 amino acids, the molecular weight of the product is 38729.40Da, and the theoretical isoelectric point is 6.56. Among them, there are 41 acidic amino acids (Asp+Glu) and 39 basic amino acids (Arg+Lys). The protein was unstable as an unstable protein, and the fat index was 80.32; the overall average hydrophilicity was -0.301.

Online analysis was performed with Phobius (http://phobius.sbc.su.se/), and the analysis results are shown in Figure 7.

As can be seen from Figure 7, the protein encoded by GA2ox may have a signal peptide. At the same time, the transmembrane domain of the GA2ox protein sequence was predicted. The results showed that the entire peptide chain was located outside the cell membrane. Combined with the prediction of the above signal peptide, it was inferred that the protein might be synthesized in the cytoplasmic matrix, and then directly anchored through protein transport. function at specific sites outside the cell membrane.

The secondary structure of GA2ox was predicted by DNAMAN, and the prediction results are shown in Figure 8.

It can be seen from Figure 8 that among the proteins encoded by GA2ox, the helical structure accounts for 21.14%, the fold accounts for 26.67%, and the random coil accounts for 52.19%.

The hydrophobicity of GA2ox was predicted by DNAMAN, and the prediction results are shown in Figure 9.

It can be seen from Figure 9 that the glutamic acid at the 91st position of the GA2ox polypeptide chain has the lowest value of -3.18, which is the most hydrophilic, while the methionine at the first position has the highest value of 2.84, and the most hydrophobicity.

In order to study the evolution of Mango GA2ox, a phylogenetic tree of the amino acid sequence of GA2ox was constructed, as shown in Figure 10.

It can be seen from Figure 10 that mango GA2ox is closely related to Ga2ox (PON72625.1), Ga2ox (PON57518.1), Ga2ox (XP_010110967.1), Ga2ox1 (ALS88210.1), Ga2ox2 (ALS88211) .1), Baklava Ga2ox3 (ALS88212.1) are closely related; followed by Castor bean Ga2ox (XP_015574955.1), Jatropha Ga2ox (NP_001292962.1), Rubber tree Ga2ox (XP_021649315.1), Cassava Ga2ox (XP_021594037. 1), rubber tree Ga2ox (XP_021676263.1).

Example 2 : Gene relative PCR detection

Beacon Designer was used to design primers to explore the expression of GA20ox gene in different periods of "Jinhuangmang" (talling characteristic) and "Guiqimang" (dwarfing characteristic).

Actin-F and actin-R primers were designed with mango actin internal reference gene. Specific primers GA20ox-YGF and GA20ox-YGR were designed according to the cloned GA20ox sequence, and the cDNA was obtained as in Example 1.

PCR quantitative reaction steps are as follows:

1) Thaw the All-in-One ^™ qPCR Mix at room temperature, mix by gentle inversion and centrifuge briefly.

2) Prepare PCR Reaction Mix on ice, including 10 μL of 2×All-in One ^TM qPCR Mix, 2 μL of qPCR Primer (1 μmol/L), 2 μL of cDNA Template, and add ddH ₂ O to make the final volume to 20 μL. In the experiment, NTC (no template control) was designed as a negative control, that is, the template cDNA was replaced by water, and other reagents remained unchanged, so as to ensure that the system was free of contamination.

3) Briefly centrifuge the reaction tube to ensure that all the reaction solution is at the bottom of the reaction tube.

The PCR reaction program was 95 °C for 10 min; 95 °C for 10 s, 60 °C for 20 s, 72 °C for 15 s, 40 cycles; 72 °C for 10 min. After the PCR reaction, the melting curve analysis was performed, that is, the temperature was controlled at 72-95 °C, increased by 0.5 °C every 10 s, and finally held at 25 °C for 30 s. Trials were set up with 3 replicates.

Data analysis was performed using 7300System software and the 2- ^ΔΔCt method, and the results are shown in Figure 11.

It can be seen from Figure 11 that in the five phenological periods, the GA20ox gene expression level of the test materials with dwarfing characteristics is much higher than that of the germplasm with arborization characteristics, indicating that the GA2ox gene has a role in regulating the dwarfing phenotype of plants.

In conclusion, the present invention can provide support for the early prediction of whether the test material has dwarfing traits by measuring the GA2ox-oxidase gene expression by semi-fluorescence quantitative analysis.

[1] SEQUENCE LISTING

[2] <110> Guangxi Zhuang Autonomous Region Subtropical Crops Research Institute

[3] <120> A gene encoding GA2ox-oxidase and its application in judging mango dwarf varieties

[4] <130> 2020

[5]

[6] <160> 2

[7]

[8] <170> PatentIn version 3.3

[9] <210> 1

[10] <211> 2075

[11] <212> DNA

[12] <213> Artificial sequences

[13]

[14] <400> 1

[15] acatggggga gagtggtact agtatagggc gattgggccc gacgtcgcat gctcccggcc 60

[16] gccatggcgg ccgcgggaat tcgattaatg gcgatgttgg atggattgaa tacctcctct120

[17] tcaccgccaa tcaagattct aatctccaac gattcgttta tcctctcgga aaaaacccgg180

[18] aaaagtttcg gtataaacct gcagaaaaaa tcattccttt tttattttcg tttgcatagc240

[19] tgtgttttat agtcttgttt ggtgttgctg cagtcgtggc aatttctgcc cgttctttcc300

[20] cttcctcaat ggcataaata ccaggcagac acacaaagct tctttatagt tagcaacgcc360

[21] tctctctgcc tctgtcttca tgcatcagca gcaaatcctc tcatttgccc atataaaccc420

[22] ttcaatctct tcagaccctt ctcaattctc accttctcct cttttccact ctcaatttct480

[23] ctctctgtct ctctctgtgt tttgtcagaa aaaactcggc caccatggtg gttctgtcac540

[24] aaccagcatt agaacatttc tctataatcg aaacttacca gccttcaagc tgcttatact600

[25] caggaattcc agttgtagac atgagacacc ctgaagccaa gttccatgta gtggaagcct660

[26] gtgaaaaata cggcttcttc aagctcatta accatgatgt tccgttggag ttcatggcca720

[27] atttagaagc cgaagctgtc aacttcttta acctccctca gtctgagaaa gacaaagctg780

[28] gaccccctga cccttatggc tatggcagca aaagcattgg ccccaatggt gatgttggtt840

[29] ggattgaata tctcctcctc aactccaacc ctcaaatcac ttcacaaaaa actctcgcca900

[30] ttttcaaaca cagccctcat gatttccgga gtgctgtgga gaagtacata acagaaatga960

[31] agaaacttgc atatgaagtt cttgaattaa tggccgatgg gctaagaata gagccaagga1020

[32] acattttcag tagattcata agggatgaaa aaagtgactc ctgtttcagg ctgaaccact1080

[33] acccaccatg tccagagctt caaacattga agaaaggaag caatttgatt gggttcggag1140

[34] aacacacaga ccctcagatt ctttctgttc taagatctaa caatacttca ggacttgaaa1200

[35] tttgcttaag agatggcact tgggtttctg tccctgctga tcattcttcc tttttcctca1260

[36] atgttggtga tgctctgcag gtaatgacta atgggagatt ccaaagtgtg aagcacagag1320

[37] tgttggctga cagagtgaaa tcaagaattt caatgatata ttttggaggg ccaccattga1380

[38] atgaaaagat tgcacctttg ccatgcctag tttcaaaaga agaagattgc ttgtacaagg1440

[39] agttcacttg gtgtgaatac aagtgctctg cctataagtc caagttggct gattataggc1500

[40] ttgggcagtt tgagaaatga aaaccaaaag gacacttccg taatttagca aaaattaaac1560

[41] tagccataca tggagaagaa tataaaatag agcctcaact tctcttgttt atttatcaac1620

[42] aactctgtac tagttaactg cagctacaat gttattatcc actagatctt gatcatgtta1680

[43] aaataaatta taattttcaa cttattttca tattttattt tatgttaacc acagtttcaa1740

[44] ttcccccttt gggtttctca ttgtcctgga atttaggcat tcaatttgag tatttgactt1800

[45] ggcatacatg cacgtcattc ttatctataa ctatcgaata tagctgccat caaaagcttt1860

[46] tgccagtacc caagcatgaa atgttttttc ctatctgtat tgatgatcaa tagagttgaa1920

[47] ttactgttcg ttaatggaga tttctgtgtt gggtggttga ttatgcaggt gctgacaaat1980

[48] ggaagattta agagcgtaag gcaaaatgtt ggagactcat tacaggtata caatttgtac2040

[49] agtttttacc aaaaaaaaaa aaaaaaaaaa aaaaa 2075

[50]

[51] <210> 2

[52] <211> 341

[53] <212> PRT

[54] <213> Artificial sequences

[55]

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[57] Met Cys Phe Val Arg Lys Asn Ser Ala Thr Met Val Val Leu Ser Gln

[58] 1 5 10 15

[59] Pro Ala Leu Glu His Phe Ser Ile Ile Glu Thr Tyr Gln Pro Ser Ser

[60] 20 25 30

[61] Cys Leu Tyr Ser Gly Ile Pro Val Val Asp Met Arg His Pro Glu Ala

[62] 35 40 45

[63] Lys Phe His Val Val Glu Ala Cys Glu Lys Tyr Gly Phe Phe Lys Leu

[64] 50 55 60

[65] Ile Asn His Asp Val Pro Leu Glu Phe Met Ala Asn Leu Glu Ala Glu

[66] 65 70 75 80

[67] Ala Val Asn Phe Phe Asn Leu Pro Gln Ser Glu Lys Asp Lys Ala Gly

[68] 85 90 95

[69] Pro Pro Asp Pro Tyr Gly Tyr Gly Ser Lys Ser Ile Gly Pro Asn Gly

[70] 100 105 110

[71] Asp Val Gly Trp Ile Glu Tyr Leu Leu Leu Asn Ser Asn Pro Gln Ile

[72] 115 120 125

[73] Thr Ser Gln Lys Thr Leu Ala Ile Phe Lys His Ser Pro His Asp Phe

[74] 130 135 140

[75] Arg Ser Ala Val Glu Lys Tyr Ile Thr Glu Met Lys Lys Leu Ala Tyr

[76] 145 150 155 160

[77] Glu Val Leu Glu Leu Met Ala Asp Gly Leu Arg Ile Glu Pro Arg Asn

[78] 165 170 175

[79] Ile Phe Ser Arg Phe Ile Arg Asp Glu Lys Ser Asp Ser Cys Phe Arg

[80] 180 185 190

[81] Leu Asn His Tyr Pro Pro Cys Pro Glu Leu Gln Thr Leu Lys Lys Gly

[82] 195 200 205

[83] Ser Asn Leu Ile Gly Phe Gly Glu His Thr Asp Pro Gln Ile Leu Ser

[84] 210 215 220

[85] Val Leu Arg Ser Asn Asn Thr Ser Gly Leu Glu Ile Cys Leu Arg Asp

[86] 225 230 235 240

[87] Gly Thr Trp Val Ser Val Pro Ala Asp His Ser Ser Phe Phe Leu Asn

[88] 245 250 255

[89] Val Gly Asp Ala Leu Gln Val Met Thr Asn Gly Arg Phe Gln Ser Val

[90] 260 265 270

[91] Lys His Arg Val Leu Ala Asp Arg Val Lys Ser Arg Ile Ser Met Ile

[92] 275 280 285

[93] Tyr Phe Gly Gly Pro Pro Leu Asn Glu Lys Ile Ala Pro Leu Pro Cys

[94] 290 295 300

[95] Leu Val Ser Lys Glu Glu Asp Cys Leu Tyr Lys Glu Phe Thr Trp Cys

[96] 305 310 315 320

[97] Glu Tyr Lys Cys Ser Ala Tyr Lys Ser Lys Leu Ala Asp Tyr Arg Leu

[98] 325 330 335

[99] Gly Gln Phe Glu Lys

[100] 340

Claims (5)

1. A gene encoding GA2ox-oxidase, wherein the nucleotide sequence of the gene is shown in SEQ ID No.1. 2. An application of the gene encoding GA2ox-oxidase according to claim 1, wherein the gene encoding GA2ox-oxidase is used to predict whether a mango variety has dwarfing characteristics. 3. A GA2ox-oxidase, wherein the GA2ox-oxidase is encoded by the gene of claim 1. 4. The GA2ox-oxidase according to claim 3, wherein the amino acid sequence of the GA2ox-oxidase is shown in SEQ ID No.2. 5 . The GA2ox-oxidase according to claim 3 , wherein the GA2ox-oxidase consists of 341 amino acids, has a molecular weight of 38729.40 Da and a theoretical isoelectric point of 6.56. 6 .

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